Oplopanax horridus (Sm.) Torr. & A. Gray ex Miq., well-known and marketed as Devil’s Club in North America, has a long history of being used medicinally for rheumatoid arthritis, autoimmune conditions, eczema, type II diabetes, external infections, and internal infections. It is also used internally as a respiratory stimulant and expectorant. The chemical constituents from the root barks are rarely studied, especially the hydrophilic interaction extracts from the root barks of O. horridus as well as qualitative and quantitative analysis of constituents from O. horridus. As a part of our research work on bioactive metabolites from root barks of O. horridus, phytochemical investigation was carefully conducted. Forty-seven compounds (OH-1~OH-47) had been isolated and purified by chromatographic methods, and their structures were identified and elucidated by spectroscopic techniques. Among these constituents, OH-9, OH-10, OH-30, OH-31, OH-33, OH-38, OH-41, OH-42, OH-44 were new compounds, and OH-47 was a new natural product. All the compounds were firstly discovered from O. horridus except OH-1~OH-6. A pressurized liquid extraction (PLE) and on-line solid phase extraction coupled with high performance liquid chromatography (SPE-HPLC) was developed for simultaneous determination of six polyynes, including falcarindiol, oplopandiol, (11S,16S,9Z)-9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate, oplopandiol acetate, oplopantriol A, oplopantriol B, and one polyene, (S,E)-nerolidol, in two species of Oplopanax, O. horridus as well as O. elatus; and GC-MS method was employed for qualitative analysis of essential oil from O. horridus, which identified fifty volatile compounds. Polyynes are regarded to be responsible for anti-cancer activities of Oplopanax horridus. Twenty-two polyynes including six natural polyynes (OH-1~OH-6) and sixteen synthesized acetylated derivatives (OHR-1~OHR-16) were prepared and their anticancer activities in MCF-7, A549, HepG2 and MM-231 cells were investigated and compared to understand their potential activity and possible structure-activity relationship